Use of decellularized extracellular matrix for encapsulating cells

ABSTRACT

A microparticle for cell encapsulation is provided, having a core which comprises continuous fibers of decellularized extracellular matrix (ECM) and, optionally, an outer layer. Also provided are methods of encapsulating cells in the microparticle, pharmaceutical compositions comprising the microparticle, and methods of treating disease in animals employing the microparticles of the invention, for example, treating Diabetes.

RELATED APPLICATIONS

This application is a National Phase of PCT Patent Application No.PCT/IL2013/050748 having International filing date of Sept. 3, 2013,which claims benefit of priority under 35 USC § 119(e) of U.S.Provisional Patent Application No. 61/696,368, filed on Sept. 4, 2012.The contents of the above applications are all incorporated by referenceas if fully set forth herein in their entirety.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates the use ofdecellularized extracellular matrix for encapsulating cells.

Cell encapsulation, a strategy whereby a pool of live cells is entrappedwithin a semipermeable membrane, represents an evolving branch ofbiotechnology and regenerative medicine.

Cell capsules are intended to protect the entrapped cell/tissuefragments against the components of the host immune system, whilesimultaneously permitting the unhindered passage of nutrients, oxygenand secreted therapeutics factors, allowing for the controlled deliveryof therapeutic products to specific physiological sites in order torestore lost function due to disease or degeneration.

Since cells are capable of secreting therapeutics such as hormones orbiological proteins in response to an external stimulus they may be usedas therapeutics in the treatment of a myriad of diseases includingendocrine disorders (diabetes, hypoparathyroidism) central nervoussystem disorders (Parkinson's and Alzheimer's), as well as conditionssuch as heart disease, and cancer. Further, cells may be engineered soas to express useful proteins, thereby increasing the range of diseasesfor which they may be used as therapeutics.

Alginates are a family of unbranched anionic polysaccharides derivedfrom brown algae (Phaeophyta) which occur extracellularly andintracellularly at approximately 20% to 40% of the dry weight. The1,4-linked α-1-guluronate (G) and β-d-mannuronate (M) are arranged inhomopolymeric (GGG blocks and MMM blocks) or heteropolymeric blockstructures (MGM blocks). Cell walls of brown algae also contain 5% to20% of fucoidan, a branched polysaccharide sulphate ester with 1-fucosefour-sulfate blocks as the major component. Commercial alginates areoften extracted from algae washed ashore, and their properties depend onthe harvesting and extraction processes.

Alginate has been employed for encapsulating cells to be transplanted,since it is biocompatible both with host and with enclosed cells;moreover, its quality can be constantly ensured. Furthermore, the use ofalginate ensures that the surface of the capsules is not rough therebypreventing the elicitation of immunological reactions when implanted.

Decellularized extracellular cell matrix (which comprises molecules suchas the collagen family (as a major macromolecule), elastic fibers,glycosoaminoglycans (GAG) and proteoglycans, and adhesive glycoproteins)has also been proposed to fabricate capsules for cell encapsulation. Thedecellularized extracellular cell matrix serves as a network supportingthe attachment and proliferation of cells.

Generation of decellularized ECM from natural tissues involvessubjecting the tissues to enzymatic cellular digestion (e.g., usingtrypsin), hypotonic, hypertonic and/or low ionic strength buffers,detergent and chemical digestion (e.g., using SDS, Triton-X-100,ammonium hydroxide, peracetic acid) and non-micellar amphipaticmolecules such as polyethylene glycole (PEG) (See for example, U.S. Pat.Appl. Nos. 20040076657, 20030014126, 20020114845, 20050191281,20050256588 and U.S. Pat. Nos. 6,933,103, 6,743,574, 6,734,018,5,855,620; and WO2006/095342 which are fully incorporated herein byreference).

Freytes et al., Biomaterials 29 (2008) 1630-1637 teaches a method ofgenerating soluble, decellularized ECM and preparation of gelstherefrom.

U.S. Patent Application No. 20120156250 teaches soluble decellularizedECM.

U.S. Patent Application No. 201000267143 teaches a scaffold ofdecellularized extracellular matrix and alginate.

U.S. Patent Application Nos. 20100189760 and 20100172942 teach amultilayered capsule for cell encapsulation, wherein at least one of thelayers comprises alginate.

Mazzitelli et al., Acta Biomaterialia 7 (2011) 1050-1062 teaches cellencapsulating particles comprising decellularized ECM and alginate.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present inventionthere is provided a microparticle for cell encapsulation, having a corewhich comprises continuous fibers of decellularized extracellular matrix(ECM).

According to an aspect of some embodiments of the present inventionthere is provided a microparticle for cell encapsulation, having a corewhich comprises continuous fibers of decellularized extracellular matrix(ECM) and an outer layer comprising a polymerizing agent, wherein anamount of polymerizing agent in the outer layer is at least 10 times theamount of polymerizing agent in the core.

According to an aspect of some embodiments of the present inventionthere is provided a microparticle for cell encapsulation having a corewhich comprises continuous fibers of decellularized extracellular matrix(ECM) and an outer layer comprising a polyion.

According to an aspect of some embodiments of the present inventionthere is provided a method of encapsulating a cell comprising:

(a) contacting solubilized decellularized ECM and cells in the presenceof a polymerizing agent to generate a mixture;

(b) generating microparticles comprising the cells; and

(c) depolymerization the polymerizing agent, thereby encapsulating thecell.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating a medical condition which maybenefit from cell transplantation in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of a plurality of the microparticles provided herein, therebytreating the medical condition.

According to an aspect of some embodiments of the present inventionthere is provided a pharmaceutical composition comprising themicroparticles provided herein as the active agent and apharmaceutically acceptable carrier.

According to an aspect of some embodiments of the present inventionthere is provided a method of treating Diabetes in a subject in needthereof, comprising administering to the subject a therapeuticallyeffective amount of a plurality of the microparticle described herein,thereby treating Diabetes.

According to some embodiments of the invention, the outer layer furthercomprises a polyion.

According to some embodiments of the invention, the microparticle has anouter layer which encapsulates the core, the outer layer comprising apolyion.

According to some embodiments of the invention, the polyion is selectedfrom the group consisting of poly-L-lysine, poly-D-lysine,poly-L,D-lysine, poly-L-omithine, poly-D-omithine, poly-L,D ornithine,chitosan, polyacrylamide, poly(vinyl alcohol), chitosan and combinationsthereof.

According to some embodiments of the invention, the outer layer furthercomprises a polymerizing agent.

According to some embodiments of the invention, the polymerizing agentis selected from the group consisting of chitosan, polymethacrylic acid,a polysaccharide, poly (ethylene glycol) (PEG) and poly (hydroxyethyl)methacrylate (HEMA).

According to some embodiments of the invention, the polysaccharide isselected from the group consisting of alginate, hyaluronic acid andagarose.

According to some embodiments of the invention, the core issubstantially devoid of the polymerizing agent.

According to some embodiments of the invention, the amount of thepolymerizing agent in the outer layer is at least ten times greater thanan amount of the polymerizing agent in the core.

According to some embodiments of the invention, the thickness of theouter layer is between about 10 μm to 30 μm.

According to some embodiments of the invention, the microparticlefurther comprises a plurality of cells.

According to some embodiments of the invention, the microparticle isbetween 300-1000 μm in diameter.

According to some embodiments of the invention, the polymerizing agentis selected from the group consisting of chitosan, polymethacrylic acid,a polysaccharide, poly (ethylene glycol) (PEG) and poly (hydroxyethyl)methacrylate (HEMA).

According to some embodiments of the invention, the polysaccharide isselected from the group consisting of alginate, hyaluronic acid andagarose.

According to some embodiments of the invention, the polysaccharide isalginate.

According to some embodiments of the invention, the depolymerizing iseffected by contacting the microparticles with a chelating agent.

According to some embodiments of the invention, the chelating agentcomprises calcium citrate or sodium citrate.

According to some embodiments of the invention, the method furthercomprises contacting the microparticles following step (b) and prior tostep (c) with a polyion to generate coated microparticles.

According to some embodiments of the invention, the polyion is selectedfrom the group consisting of poly-L-lysine, poly-D-lysine,poly-L,D-lysine, poly-L-omithine, poly-D-omithine, poly-L,D ornithine,chitosan, polyacrylamide, poly(vinyl alcohol), chitosan and combinationsthereof.

According to some embodiments of the invention, the generating themicroparticles is effected by extruding the mixture into a crosslinkingsolution.

According to some embodiments of the invention, the crosslinkingsolution comprises calcium chloride or a strontium solution.

According to some embodiments of the invention, the decellularizedextracellular matrix is derived from pancreatic tissue.

According to some embodiments of the invention, the decellularizedextracellular matrix is derived from cardiac tissue.

According to some embodiments of the invention, the microparticlefurther comprises cells which express pancreatic beta cell markers.

According to some embodiments of the invention, the microparticlefurther comprises cells which express cardiac markers.

Unless otherwise defined, all technical and/or scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which the invention pertains. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of embodiments of the invention, exemplarymethods and/or materials are described below. In case of conflict, thepatent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and are notintended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this paten or patent application publication with colordrawings(s) will be provided by the Office upon request and payment ofthe necessary fee.

Some embodiments of the invention are herein described, by way ofexample only, with reference to the accompanying drawings. With specificreference now to the drawings in detail, it is stressed that theparticulars shown are by way of example and for purposes of illustrativediscussion of embodiments of the invention. In this regard, thedescription taken with the drawings makes apparent to those skilled inthe art how embodiments of the invention may be practiced.

In the drawings:

FIG. 1 is a pictorial representation of the overall process ofpreparation of capsules of ECM according to embodiments of the presentinvention.

FIG. 2 is a pictorial representation of the process of liquification andejection of the alginate from the ECM-cell capsules coated with a PLLmembrane.

FIGS. 3A-F are photographs illustrating the characterization of thedecellularized pancreatic ECM. (A) Native porcine pancreas. (B)Decellularization of native porcine pancreas. (C) Sections ofdecellularized pancreas were stained with H&E staining in order toassure that all cellular components which can provoke immune responseare removed. As seen, no traces or residues of cellular or nuclearcomponents could be detected in the decellularized pancreas. (D) HR-SEMimage exhibited typical morphology of collagen fibers. (E) LyophilizedECM. (F) Soluble ECM.

FIGS. 4A-B illustrate proteomic and rheological analysis of pancreaticECM gel. (A) A proteomic analysis of the ECM was performed and comparedto one performed on native pancreas and solubilized decellularizedpancreatic matrix. Samples were digested with trypsin, analyzed byLC-MS/Mson LTQ-Orbitrap (Thermo) and identified by Mascot and Seguessoftware against the mammalian part of the NCBI-nr database, and a decoydata base (in order to determine the false discovery rate). The samemain proteins were detected in the decellularized pancreatic ECM andsolubilized decellularized pancreatic matrix. Thus, it can be deducedthat the acellularization process of the pancreatic ECM does not damagethose proteins. Collagen 1 was significantly present in all samples. (B)Representative curve of the gelation kinetics of pancreatic gelsdetermined during the mechanical testing of the gels based on thestorage modulus (G′) and the loss modulus (G″).

FIGS. 5A-K illustrate the characterization and therapeutic efficacy ofcells seeded on ECM gels. (A-C) 3D structure (SEM) of reconstitutedliquefied ECM with (D-F) viable cells on it (WETSEM). Confocal imagingof (G) cells seeded on plate and (H) cells stained with Hoechst,phalloidin and (I) DIL. Secretion of C-peptide from (J) hMSC and (K) HumHep seeded on tissue culture plate (TCP) or gel, 5 days post viraltransduction (***p<0.001). n=7.

FIGS. 6A-D illustrate characterization of Alginate-PLL microcapsules.(A) Encapsulated cells viability was determined using the Alamar Blueassay. It was found that both kinds of cells, Hum Hep cells and hMSC,were viable for more than 120 days. (B) Fluorescent micrographs ofalginate-PLL FITC capsules and (C) encapsulated cells stained withFluorescein Diacetate (FDA) cell viability assay were taken on day 10and (D) 108 post encapsulation.

FIGS. 7A-G illustrate characterization and therapeutic efficacy ofencapsulated cells. Encapsulated cells were stained with (A) Hoechst and(B) FDA. (C) Collagen fibers were stained with Tamra (red). (D) Doublestaining with Tamra+FDA (orange). Secretion of C-peptide from (E) HumHep cells and (F,G) hMSC 5 days post viral transduction (**p<0.01,***p<0.001). n=7.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to the useof decellularized extracellular matrix for encapsulating cells.

Encapsulating cells in hydrogels has shown promising results forreducing the immune response, but many preclinical and clinical trialsresults have been inconsistent because of the limited ability ofhydrogels to support cellular viability and function over an extendedtime period. Therefore, few successful products have been fullycommercialized based on these cell-encapsulation technologies. Mixingcells and polymer solutions during a cell encapsulation processgenerally leads to a significant decrease in cell viability, becausehigh shear stresses can disrupt cell-cell contacts.

The present inventors have now conceived a way to protect cells duringthe encapsulation process by mixing them with solubilized decellularizedextracellular matrix. In order to ensure that smooth capsules aregenerated, a polymerizing agent such as alginate is used during theencapsulation process. The polymerizing agent may subsequently beremoved from the inner core of the capsules by subsequentdepolymerization.

Thus, according to one aspect of the present invention, a method ofencapsulating a cell is provided. The method comprises:

(a) contacting solubilized decellularized extracellular matrix (ECM) andcells in the presence of a polymerizing agent to generate a mixture;

(b) extruding the mixture into a crosslinking solution to generatemicroparticles comprising the cells;

(c) contacting the microparticles with a chelating agent underconditions which allow depolymerization of the polymerizing agent,thereby encapsulating the cell.

As used herein the phrase “decellularized ECM of a tissue” refers to theextracellular matrix which supports tissue organization (e.g., a naturaltissue) and underwent a decellularization process (i.e., a removal ofall cells from the tissue) and is thus completely devoid of any cellularcomponents.

The phrase “completely devoid of any cellular components” as used hereinrefers to being more than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, (e.g., 100%) devoid of the cellular components present in thenatural (e.g., native) tissue. As used herein, the phrase “cellularcomponents” refers to cell membrane components or intracellularcomponents which make up the cell. Examples of cell components includecell structures (e.g., organelles) or molecules comprised in same.Examples of such include, but are not limited to, cell nuclei, nucleicacids, residual nucleic acids (e.g., fragmented nucleic acid sequences),cell membranes and/or residual cell membranes (e.g., fragmentedmembranes) which are present in cells of the tissue. It will beappreciated that due to the removal of all cellular components from thetissue, such a decellularized matrix cannot induce an immunologicalresponse when implanted in a subject.

The phrase “extracellular matrix (ECM)” as used herein, refers to acomplex network of materials produced and secreted by the cells of thetissue into the surrounding extracellular space and/or medium and whichtypically together with the cells of the tissue impart the tissue itsmechanical and structural properties. Generally, the ECM includesfibrous elements (particularly collagen, elastin, or reticulin), celladhesion polypeptides (e.g., fibronectin, laminin and adhesiveglycoproteins), and space-filling molecules [usually glycosaminoglycans(GAG), proteoglycans].

A tissue-of-interest (e.g., pancreas, myocardium) may be derived from anautologous or non-autologous tissue (e.g., allogeneic or even xenogeneictissue, due to non-immunogenicity of the resultant decellularizedmatrix). The tissue is removed from the subject [e.g., an animal,preferably a mammal, such as a pig, monkey or chimpanzee, oralternatively, a deceased human being (shortly after death)] and washede.g. in a sterile saline solution (0.9% NaCl, pH=7.4) or phosphatebuffered saline (PBS), which can be supplemented with antibiotics suchas Penicillin/Streptomycin 250 units/ml. Although whole tissues can beused, for several applications segments of tissues may be cut e.g.sliced. Such tissue segments can be of various dimensions, depending onthe original tissue used and the desired application.

To remove the vasculature surrounding and feeding the tissue, the tissuemay be washed at room temperature by agitation in large amounts (e.g.,50 ml per each gram of tissue segment) of EDTA solution (0.5-10 mM,pH-7.4).

Next, the tissue is subjected to a hypertonic or hypotonic buffer tothereby obtain increased intercellular space within the tissue.

The hypertonic buffer used by the present invention can be any buffer orsolution with a concentration of solutes that is higher than thatpresent in the cytoplasm and/or the intercellular liquid within thetissue [e.g., a concentration of NaCl which is higher than 0.9% (w/v)].Due to osmosis, incubation of the tissue with the hypertonic bufferresults in increased intercellular space within the tissue.

According to another embodiment, peracetic acid is used to decellularizethe tissue.

Preferably, the hypertonic buffer used by the method according to thisaspect of the present invention includes sodium chloride (NaCl) at aconcentration which is higher than 0.9% (w/v), preferably, higher than1% (w/v), preferably, in the range of 1-1.2% (w/v), e.g., 1.1% (w/v).

Preferably, the hypotonic buffer used by the method according to thisaspect of the present invention includes sodium chloride (NaCl) at aconcentration which is lower than 0.9% (w/v), lower than 0.8% (w/v),lower than 0.7% (w/v), preferably, in the range of 0.6-0.9% (w/v), e.g.,0.7% (w/v).

According to this aspect of the present invention, the tissue issubjected to the hypertonic or hypotonic buffer for a time periodleading to the biological effect, i.e., cell shrinkage which leads toincreased intercellular space within the tissue.

According to a particular embodiment, the tissue is contacted with ahypertonic buffer (e.g. 1.1% w/v) and subsequently contacted with ahypotonic buffer (e.g. 0.7% w/v). This procedure may be repeated for twoor more cycles.

Preferably, the hypotonic buffer used by the method according to thisaspect of the present invention includes sodium chloride (NaCl) at aconcentration which is lower than 0.9% (w/v), lower than 0.8% (w/v),lower than 0.7% (w/v), preferably, in the range of 0.6-0.9% (w/v), e.g.,0.7% (w/v).

Following incubation with the hypertonic/hypotonic buffer, the tissue isfurther subjected to an enzymatic proteolytic digestion which digestsall cellular components within the tissue yet preserves the ECMcomponents (e.g., collagen and elastin) and thus results in a matrixwhich exhibits the mechanical and structural properties of the originaltissue ECM. It will be appreciated that measures are taken to preservethe ECM components while digesting the cellular components of thetissue. These measures are further described hereinbelow and include,for example, adjusting the concentration of the active ingredient (e.g.,trypsin) within the digestion solution as well as the incubation time.

Proteolytic digestion according to this aspect of the present inventioncan be effected using a variety of proteolytic enzymes. Non-limitingexamples of suitable proteolytic enzymes include trypsin and pancreatinwhich are available from various sources such as from Sigma (St Louis,Mo., USA). According to one preferred embodiment of this aspect of thepresent invention, proteolytic digestion is effected using trypsin.

Digestion with trypsin is preferably effected at a trypsin concentrationranging from 0.01-0.25% (w/v), more preferably, 0.02-0.2% (w/v), morepreferably, 0.05-0.1 (w/v), even more preferably, a trypsinconcentration of about 0.05% (w/v). For example, a trypsin solutioncontaining 0.05% trypsin (w/v; Sigma), 0.02% EDTA (w/v) and antibiotics(Penicillin/Streptomycin 250 units/ml), pH=7.2] may be used toefficiently digest all cellular components of the tissue.

It will be appreciated that for efficient digestion of all cellularcomponents of the tissue, each of the tissue segments may be placed in aseparate vessel containing the digestion solution (e.g., a trypsinsolution as described hereinabove) in a ratio of 40 ml digestionsolution per each gram of tissue. Preferably, while in the digestionsolution, the tissue segments are slowly agitated (e.g., at about 150rpm) to enable complete penetration of the digestion solution to allcells of the tissue.

It should be noted that the concentration of the digestion solution andthe incubation time therein depend on the type of tissue being treatedand the size of tissue segments utilized and those of skilled in the artare capable of adjusting the conditions according to the desired sizeand type of tissue.

Preferably, the tissue segments are incubated for at least about 20hours, more preferably, at least about 24 hours. Preferably, thedigestion solution is replaced at least once such that the overallincubation time in the digestion solution is at least 40-48 hours.

Next, the cellular components are removed from the tissue. Removal ofthe digested components from the tissue can be effected using variouswash solutions, such as detergent solutions (e.g., ionic and non ionicdetergents such as SDS Triton X-100, Tween-20, Tween-80) which can beobtained from e.g., Sigma (St Louis, Mo., USA) or Biolab (Atarot,Israel, Merck Germany).

Preferably, the detergent solution used by the method according to thisaspect of the present invention includes TRITON-X-100 (available fromMerck). For efficient removal of all digested cellular components,TRITON-X-100 is provided at a concentration range of 0.05-2.5% (v/v),more preferably, at 0.05-2% (v/v), more preferably at 0.1-2% (v/v), evenmore preferably at a concentration of 1% (v/v).

Preferably, for optimized results, the detergent solution includes alsoammonium hydroxide, which together with the TRITON-X-100, assists inbreaking and dissolving cell nuclei, skeletal proteins, and membranes.

Preferably, ammonium hydroxide is provided at a concentration of0.05-1.5% (v/v), more preferably, at a concentration of 0.05-1% (v/v),even more preferably, at a concentration of 0.1-1% (v/v) (e.g., 0.1%).

The concentrations of TRITON-X-100 and ammonium hydroxide in thedetergent solution may vary, depending on the type and size of tissuebeing treated and those of skills in the art are capable of adjustingsuch concentration according to the tissue used.

Incubation of the tissue (or tissue segments) with the detergentsolution can last from a few minutes to hours to even several days,depending on the type and size of tissue and the concentration of thedetergent solution used and those of skills in the art are capable ofadjusting such incubation periods. Preferably, incubation with thedetergent solution is effected for at least 24-72 hours. According toone embodiment, 2-4 cycles of incubation with the detergent solution areperformed until no foam is observed, such that the total incubation timemay be between about 150-200 hours.

Although as described hereinabove, incubation with the detergentsolution enables the removal of cell nuclei, proteins and membrane, themethod according to this aspect of the present invention optionally andpreferably includes an additional step of removing nucleic acids (aswell as residual nucleic acids) from the tissue to thereby obtain anucleic acid—free tissue. As used herein the phrase “nucleic acid—freetissue” refers to a tissue being more than 99% free of any nucleic acidor fragments thereof as determined using conventional methods (e.g.,spectrophotometry, electrophoresis essentially as described in Example 1of the Examples section which follows). Such a step utilizes a DNasesolution (and optionally also an RNase solution). Suitable nucleasesinclude DNase and/or RNase [Sigma, Bet Haemek Israel, 20 μg/ml in Hankbalance salt solution (HBSS)].

The above described detergent solution is preferably removed bysubjecting the matrix to several washes in water or saline (e.g., atleast 10 washes of 30 minutes each, and 2-3 washes of 24 hours each),until there is no evident of detergent solution in the matrix.

Optionally, the decellularized ECM is then sterilized. Sterilization ofthe decellularized ECM may be effected using methods known in the art(e.g. 70% ethanol).

Solubilization of the decellularized ECM may be effected as described inFreytes et al., Biomaterials 29 (2008) 1630-1637 and U.S. PatentApplication No. 20120156250, the contents of which are incorporatedherein by reference.

Typically, in order to carry out solubilization of the decellularizedECM it is first lyophilized.

The lyophilized decellularized is typically cut into small pieces, e.g.crumbled and then subjected to a second round of proteolytic digestion.The digestion is effected under conditions that allow the proteolyticenzyme to digest and solubilize the ECM. Thus, according to oneembodiment, the digestion is carried out in the presence of an acid(e.g. HCL) so as to obtain a pH of about 3-4.

Proteolytic digestion according to this aspect of the present inventioncan be effected using a variety of proteolytic enzymes. Non-limitingexamples of suitable proteolytic enzymes include trypsin, pepsin,collaganease and pancreatin which are available from various sourcessuch as from Sigma (St Louis, Mo., USA) and combinations thereof. Matrixdegrading enzymes such as matrix metalloproteinases are alsocontemplated.

It should be noted that the concentration of the digestion solution andthe incubation time therein depend on the type of tissue being treatedand the size of tissue segments utilized and those of skilled in the artare capable of adjusting the conditions according to the desired sizeand type of tissue.

Preferably, the tissue segments are incubated for at least about 20hours, more preferably, at least about 24 hours. Preferably, thedigestion solution is replaced at least once such that the overallincubation time in the digestion solution is at least 40-48 hours.

Once the decellularized ECM is solubilized, the pH of the solution isincreased so as to irreversibly inactivate the proteolytic enzyme (e.g.to about pH 7). The decellularized ECM may be stored at this stage attemperatures lower than 20° C.—for example 4° C. so that thedecellularized ECM remains in solution.

As mentioned, the solubilized decellularized extracellular matrix (ECM)is contacted with a polymerizing agent to generate a mixture forgenerating capsules.

The polymerizing agent of this aspect of the present invention ispreferably water soluble and may include polymers such as chitosan andpolymethacrylic acid or hydrogels composed of polysaccharides (such asalginate, hyaluronic acid and agarose) or other polymers such as polyethylene glycol, (PEG), and poly hydroxyethyl methacrylate (HEMA)).

According to a particular embodiment, the polymerizing agent is chitosanor alginate.

According to another embodiment, the polymerizing agent is alginate.Alginate is commercially available from a variety of sources—e.g.Novamatrix, Norway. The alginate may be of a viscosity less than 20 upuntil greater than 200 mPa·s with different G/M content (e.g. from lessthan 1 to greater than 1.5).

Typical ratios of volumes of polymerizing agent: decellularized ECMwhich are mixed to generate the mixture are between 50:50-70:30.

Cells are added to the above described mixture. Thus, for example for a2 ml mixture, about two million cells may be added.

The present invention contemplates encapsulating any type of cell,including for example primary cells, cultured cells, single cellsuspensions of cells, clusters of cells e.g. islets, cells which arecomprised in tissues and/or organs etc.

The cells may be derived from any organism including for examplemammalian cells, (e.g. human), plant cells, algae cells, fungal cells(e.g. yeast cells), prokaryotic cells (e.g. bacterial cells).

According to a particular embodiment the cells comprise stem cells—e.g.adult stem cells such as mesenchymal stem cells or pluripotent stemcells such as embryonic stem cells or induced pluripotent stem cells.The stem cells may be modified so as to undergo ex vivo differentiation.

According to a particular embodiment, the cells are preferably intact(i.e. whole), and preferably viable, although it will be appreciatedthat pre-treatment of cells, such as generation of cell extracts ornon-intact cells are also contemplated by the present invention.

The cells may be fresh, frozen or preserved in any other way known inthe art (e.g. cryopreserved).

According to another embodiment, the cells are derived from the pancreasor the liver.

The tissue from which the decellularized extracellular matrix isproduced may be selected (i.e. matched) according to the cells which areincorporated therein.

Thus, for example when the cells are derived from the pancreas—e.g.pancreatic beta cells (or modified so as to imitate pancreatic betacells), according to certain embodiments, the tissue from which thedecellularized extracellular matrix is produced is pancreatic tissue.

In a similar fashion, when the cells are derived from cardiactissue—e.g. cardiac myocardial cells (or modified so as to imitatecardiac myocardial cells), according to certain embodiments, the tissuefrom which the decellularized extracellular matrix is produced iscardiac myocardial tissue.

Typically, the cells secrete a factor (e.g. a polypeptide) that isuseful for the treatment of a disease.

Such factors include for example, hormones including but not limited toinsulin, thyroxine, growth hormone, testosterone, oestrogen,erythropoietin and aldosterone; enzymes, including but not limited tolysosomal enzyme such as glucocerebrosidase (GCD), acidsphingomyelinase, hexosaminidase, α-N-acetylgalactosaminidise, acidlipase, α-galactosidase, α-L-iduronidase, iduronate sulfatase,α-mannosidase, sialidase, α fucosidase, G_(M1)-β-galactosidase, ceramidelactosidase, arylsulfatase A, β galactosidase and ceramidase; clottingfactors such as factor VIII.

According to a preferred embodiment, the cells secrete insulin.

As used herein, the term “insulin” refers to an insulin obtained bysynthesis or recombination, in which the peptide sequence is thesequence of human insulin, includes the allelic variations and thehomologs. The polypeptide sequence of the insulin may be modified toimprove the function of the insulin (e.g. long lasting).

Cells which secrete neurotrophic factors are also contemplated by thepresent invention.

As used herein, the phrase “neurotrophic factor” refers to a cell factorthat acts on the cerebral nervous system comprising growth,differentiation, functional maintenance and/or survival effects onneurons. Examples of neurotrophic factors include, but are not limitedto, glial derived neurotrophic factor (GDNF), GenBank accession nos.L19063, L15306; brain-derived neurotrophic factor (BDNF), GenBankaccession no CAA62632; neurotrophin-3 (NT-3), GenBank Accession No.M37763; neurotrophin-4/5; Neurturin (NTN), GenBank Accession No.NP_004549; Neurotrophin-4, GenBank Accession No. M86528; Persephin,GenBank accession No. AAC39640; brain derived neurotrophic factor,(BDNF), GenBank accession No. CAA42761; artemin (ART), GenBank accessionNo. AAD13110; ciliary neurotrophic factor (CNTF), GenBank accession No.NP_000605; insulin growth factor-I (IGF-1), GenBank accession No.NP_000609; and Neublastin GenBank accession No. AAD21075.

Cells which secrete neuropeptides are also contemplated by the presentinvention. Examples of neuropeptides include, but are not limited toOxytocin, Vasopressin, Corticotropin releasing hormone (CRH), Growthhormone releasing hormone (GHRH), Luteinizing hormone releasing hormone(LHRH), Somatostatin growth hormone release inhibiting hormone,Thyrotropin releasing hormone (TRH), Neurokinin α (substance K),Neurokinin β, Neuropeptide K, Substance P, β-endorphin, Dynorphin, Met-and leu-enkephalin, Neuropeptide tyrosine (NPY), Pancreatic polypeptide,Peptide tyrosine-tyrosine (PYY), Glucogen-like peptide-1 (GLP-1),Peptide histidine isoleucine (PHI), Pituitary adenylate cyclaseactivating peptide (PACAP), Vasoactive intestinal polypeptide (VIP),Brain natriuretic peptide, Calcitonin gene-related peptide (CGRP) (α-and β-form), Cholecystokinin (CCK), Galanin, Islet amyloid polypeptide(IAPP), Melanin concentrating hormone (MCH), ACTH, α-MSH, NeuropeptideFF, Neurotensin, Parathyroid hormone related protein, Agoutigene-related protein (AGRP), Cocaine and amphetamine regulatedtranscript (CART)/peptide, Endomorphin-1 and -2,5-HT-moduline,Hypocretins/orexins Nociceptin/orphanin FQ, Nocistatin, Prolactinreleasing peptide, Secretoneurin and Urocortin.

Cells which secrete neurotransmitters are also contemplated by thepresent invention.

A neurotransmitter according to the teaching of the present inventioncan be any substances which is released on excitation from the axonterminal of a presynaptic neuron of the central or peripheral nervoussystem and travel across the synaptic cleft to either excite or inhibitthe target cell. The neurotransmitter can be, for example, dopamine,norepinephrine, epinephrine, gamma aminobutyric acid, serotonin,acetylcholine, glycine, histamine, vasopressin, oxytocin, a tachykinin,cholecytokinin (CCK), neuropeptide Y (NPY), neurotensin, somatostatin,an opioid peptide, a purine or glutamic acid.

According to one embodiment, the cells are naïve (non-geneticallymodified).

The present invention also contemplates use of cells which have beengenetically modified to express a recombinant protein. The recombinantprotein may be a therapeutic protein or may promote in vivo longevity(AM, adrenomedullin, Jun-Ichiro et al. Tissue Eng. 2006) or may promoteneurotransmitter release (e.g., such as by transfecting with tyrosinehydroxylase).

Examples of therapeutic, recombinant proteins that may be expressed inthe cells of the present invention include, but are not limited to anantibody, insulin, human growth hormone (rHGH), follicle stimulatinghormone, factor VIII, erythropoietin, Granulocyte colony-stimulatingfactor (G-CSF),alpha-glactosidase A, alpha-L-iduronidase (rhIDU;laronidase), N-acetylgalactosamine-4-sulfatase (rhASB; galsulfase)Tissue plasminogen activator (TPA), Glucocerebrosidase, Interferon (IF)Interferon-beta-1a, Interferon beta-1b, Insulin-like growth factor 1(IGF-1), somatotropin (ST) and chymosin.

Other examples of exogenous polynucleotides which may be expressed inaccordance with the present teachings include, but are not limited to,polypeptides such as peptide hormones, antibodies or antibody fragments(e.g., Fab), enzymes and structural proteins or dsRNA,antisense/ribozyme transcripts which can be directed at specific targetsequences (e.g., transcripts of tumor associated genes) to therebydownregulate activity thereof and exert a therapeutic effect. Similarly,protective protein antigens for vaccination (see, for example, Babiuk Set al J Control Release 2000; 66:199-214) and enzymes such asfibrinolysin for treatment of ischemic damage (U.S. Pat. No. 5,078,995to Hunter et al) may expressed in the cells for transdermal ortranscutaneous delivery. The therapeutic protein can also be a prodrug.

Methods of expressing exogenous polynucleotides in cells are well knownin the art.

As used herein, the term “expressed” when used in context with theexogenous polynucleotide refers to generation of a polynucleotide(transcript) or a polypeptide product.

An integrative or episomal nucleic acid expression construct may beemployed.

Thus, the expression construct can be designed as a gene knock-inconstruct in which case it will lead to genomic integration of constructsequences, or it can be designed as an episomal expression vector.

In any case, the expression construct can be generated using standardligation and restriction techniques, which are well known in the art(see Maniatis et al., in: Molecular Cloning: A Laboratory Manual, ColdSpring Harbor Laboratory, New York, 1982). Isolated plasmids, DNAsequences, or synthesized oligonucleotides are cleaved, tailored, andreligated in the form desired.

At its minimum, the expression vector of the present invention comprisesa polynucleotide encoding the gene of interest.

The expression vector of the present invention may also includeadditional sequences which render this vector suitable for replicationand integration in prokaryotes, eukaryotes, or preferably both (e.g.,shuttle vectors) and ultimately in the cells described herein. Typicalcloning vectors contain transcription and translation initiationsequences (e.g., promoters, enhancers) and transcription and translationterminators (e.g., polyadenylation signals).

In addition to the elements already described, the expression vector ofthe present invention may contain other specialized elements intended toincrease the level of expression of cloned nucleic acids or tofacilitate the identification of cells that carry the recombinant DNA.For example, a number of animal viruses contain DNA sequences thatpromote the extra chromosomal replication of the viral genome inpermissive cell types. Plasmids bearing these viral replicons arereplicated episomally as long as the appropriate factors are provided bygenes either carried on the plasmid or with the genome of the host cell.

The vector may or may not include a eukaryotic replicon. If a eukaryoticreplicon is present, then the vector is amplifiable in eukaryotic cellsusing the appropriate selectable marker. If the vector does not comprisea eukaryotic replicon, no episomal amplification is possible. Instead,the recombinant DNA integrates into the genome of the engineered cell,where the promoter directs expression of the desired nucleic acid.

Examples of mammalian expression vectors include, but are not limitedto, pcDNA3, pcDNA3.1(+/−), pGL3, pZeoSV2(+/−), pSecTag2, pDisplay,pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1,pNMT41, pNMT81, which are available from Invitrogen, pCI which isavailable from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which areavailable from Strategene, pTRES which is available from Clontech, andtheir derivatives.

Expression vectors containing regulatory elements from eukaryoticviruses such as retroviruses can be also used. SV40 vectors includepSVT7 and pMT2. Vectors derived from bovine papilloma virus includepBV-1MTHA, and vectors derived from Epstein Bar virus include pHEBO, andp2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMTO10/A⁺,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the SV-40 early promoter, SV-40 laterpromoter, metallothionein promoter, murine mammary tumor virus promoter,Rous sarcoma virus promoter, polyhedrin promoter, or other promotersshown effective for expression in eukaryotic cells.

Recombinant viral vectors may also be used to transduce (i.e. infect)the cells of the present invention. Viruses are very specializedinfectious agents that have evolved, in many cases, to elude hostdefense mechanisms. Typically, viruses infect and propagate in specificcell types. The targeting specificity of viral vectors utilizes itsnatural specificity to specifically target predetermined cell types andthereby introduce a recombinant gene into the infected cell.

Retroviral constructs of the present invention may contain retroviralLTRs, packaging signals, and any other sequences that facilitatecreation of infectious retroviral vectors. Retroviral LTRs and packagingsignals allow the polypeptides of the invention to be packaged intoinfectious particles and delivered to the cell by viral infection.Methods for making recombinant retroviral vectors are well known in theart (see for example, Brenner et al., PNAS 86:5517-5512 (1989); Xiong etal., Developmental Dynamics 212:181-197 (1998) and references therein;each incorporated herein by reference).

Examples of retroviral sequences useful in the present invention includethose derived from adenovirus, lentivirus, Herpes simplex I virus, oradeno-associated virus (AAV). Other viruses known in the art are alsouseful in the present invention and therefore will be familiar to theordinarily skilled artisan.

Various methods can be used to introduce the expression vector of thepresent invention into cells. Such methods are generally described inSambrook et al., Molecular Cloning: A Laboratory Manual, Cold SpringsHarbor Laboratory, New York (1989, 1992), in Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley and Sons, Baltimore, Md.(1989), Chang et al., Somatic Gene Therapy, CRC Press, Ann Arbor, Mich.(1995), Vega et al., Gene Targeting, CRC Press, Ann Arbor Mich. (1995),Vectors: A Survey of Molecular Cloning Vectors and Their Uses,Butterworths, Boston Mass. (1988) and Gilboa et at. [Biotechniques 4(6): 504-512, 1986] and include, for example, stable or transienttransfection, lipofection, electroporation and infection withrecombinant viral vectors. In addition, see U.S. Pat. Nos. 5,464,764 and5,487,992 for positive-negative selection methods.

Once the mixture of polymerizing agent, soluble decellularized ECM andcells is obtained, microparticles are generated.

Typical diameters of the microparticles are between 100-1500 μm, morepreferably between 300-1000 μm.

According to one embodiment, the mixture is extruded into a crosslinkingsolution through an aperture to generate microparticles.

As used herein, the term “extruding” as used herein refers to theforcing of a flowable material out through a relatively narrow aperture(i.e. a nozzle in the widest sense), e.g. through a needle.

According to one embodiment the aperture has an inner diameter of about10-100 gauge (ga) (e.g. about 21 ga).

Typical flow rate of extrusion are about 5-10 ml/min—e.g. 6.7 ml/min.The size of the falling droplets and thus the size of the microparticlesgenerated may be regulated by altering the droplet falling distance.Exemplary droplet falling distances are between 2-5 cm—e.g. 3.5 cm.

Exemplary crosslinking solutions contemplated by the present inventioninclude for example calcium chloride (e.g. 1.5%) or strontium inbuffered solutions (e.g. PBS or HBS).

Another way of generating microparticles is by emulsification withparaffin oil. This may be particularly effective when using agarose asthe polymerizing agent. To induce gelation of the agarose, the mixtureis cooled and the oil phase is removed by suction.

The microparticles are then liquefied by subjecting the microparticlesto depolymerizing conditions (e.g. adding a depolymerizing agent). Suchconditions allow the polymerizing agent to dissolve and diffuse from theparticles.

The precise method selected for depolymerizing the polymerizing agentwill depend of the polymerizing agent used.

Examples of depolymerizing agents useful for depolymerizing alginateinclude sodium citrate (1.5%), HEPES and EDTA. The contacting time forthe depolymerizing agent is typically between 1 minute and one hour—e.g.five minutes.

Chitosan may be dissolved by lowering the pH to about 6.

The present invention further contemplates coating the microspheres witha polyion prior to the depolymerizing step and following thecrosslinking step.

Preferably, the polyion is selected from the group consisting ofpoly-L-lysine, poly-D-lysine, poly-L,D-lysine, polyethylenimine,polyallylamine, poly-L-omithine, poly-D-omithine, poly-L,D-ornithine,polyL-aspartic acid, poly-D-aspartic acid, poly-L,D-aspartic acid,polyacrylic acid, poly-L-glutamic acid, poly-D-glutamic acid,poly-L,D-glutamic acid, succinylated poly-L-lysine, succinylatedpoly-D-lysine, succinylated poly-L,D lysine, chitosan, polyacrylamide,poly(vinyl alcohol) and combinations thereof. More preferably, thepolyion is a polycation selected from the group consisting ofpoly-L-lysine, poly-D-lysine, poly-L,D-lysine, poly-L-ornithine,poly-D-ornithine, poly-L,D ornithine, chitosan, polyacrylamide,poly(vinyl alcohol), and combinations thereof. Most preferably, thepolyion is poly-L-lysine.

According to another embodiment, the polyion is chitosan.

Using the methods described herein, microparticles are obtained withnovel characteristics.

Thus, according to another aspect of the present invention there isprovided a microparticle for cell encapsulation, having a core whichcomprises continuous fibers of decellularized extracellular matrix(ECM).

The term “microparticle” refers to a particle being of microndimensions. The microparticles may be of any shape, including, withoutlimitation, elongated particle shapes, such as nanowires, or irregularshapes, in addition to more regular shapes, such as generally spherical,hexagonal and cubic microparticles. According to one embodiment, themicroparticles are generally spherical.

Rounded particles are typically characterized quantitatively by ageometrical quantity known as sphericity, which generally quantifies thedeviation of a particular geometrical shape from a perfect sphere.

Ideally, the sphericity of a three dimensional object is calculated bydividing the volume of the object to the volume of a spherecircumscribing the object. However, for some objects, the determinationof the volume is difficult and oftentimes impossible. Therefore, forpractical reasons, an alternative “two-dimensional” definition ofsphericity is used. According to this alternative, the sphericity isdefined as the ratio between the area of the projection of the objectonto a certain reference plane and the area of a circle circumscribingthe projection. For example, suppose that an image of the object isdisplayed on a planar display, then the planar display can be consideredas a reference plane and the image of the object can be considered asthe projection of the object on the reference plane.

Thus, denoting the area of the image by A and the perimeter of the imageby P, the sphericity, s, can be defined as s=4πcA/P². As will beappreciated by one of ordinary skill in the art, when the image is aperfect circle, A=π(P/2π)²=P²/4π and s=1. When the area of the image is0 (i.e., the image is a line or a curve) s=0.

Unless otherwise defined, “sphericity,” as used herein, refers totwo-dimensional sphericity.

It is recognized that the “two dimensional” sphericity is, to a goodapproximation, equivalent to the “three dimensional” sphericity (ratioof volumes), provided it is calculated and averaged over many particles(say 10 or more) or many different reference planes. In such event,starting from the “two dimensional” sphericity, s, the “threedimensional” sphericity can be defined as the cubic root of s².

According to a preferred embodiment of the present invention thesphericity of the particle is at least 80% more preferably at least 85%.

The microparticles generated according to the method of the presentinvention are substantially homogeneous, i.e. are all of a uniform shapeand size. According to one embodiment the microparticle population doesnot comprise microparticles which differ by more than 5%, 10%, 20% or30% from the size of the average microparticle in the population.

As used herein, the phrase “a core which comprises continuous fibers ofdecellularized ECM” refers to a core wherein the ECM fibers aredistributed homogenously throughout the core and are not present asclusters or particles.

According to one embodiment, the core is essentially devoid ofpolymerizing agent (e.g. alginate).

The microparticles may comprise an outer layer encapsulating the innercore. Such a layer may be fabricated from a polyion, as furtherdescribed herein above. Typically, an amount of polymerizing agent inouter layer is at least 10 times, at least 20 times, at least 50 times,at least 100 times the amount of polymerizing agent in the core. Thethickness of the outer layer is typically between 5 μm to 50 μm—e.g.between 10 μm to 30 μm.

The microparticles may be stored at temperatures above 25° C. (e.g.about 37° C.) in order to enhance the solidification of the particlesare aid in maintenance of the structural integrity of themicroparticles.

Encapsulated cells generated according to the present teachings can beused in a myriad of research and clinical applications.

Thus, according to another aspect of the present invention there isprovided a method of transplanting encapsulated cells such as fortreating a medical condition (e.g., pathology, disease, syndrome) whichmay benefit from cell transplantation in a subject in need thereof.

As used herein the term “treating” refers to inhibiting or arresting thedevelopment of a pathology and/or causing the reduction, remission, orregression of a pathology. Those of skill in the art will understandthat various methodologies and assays can be used to assess thedevelopment of a pathology, and similarly, various methodologies andassays may be used to assess the reduction, remission or regression of apathology. Preferably, the term “treating” refers to alleviating ordiminishing a symptom associated with a cancerous disease. Preferably,treating cures, e.g., substantially eliminates, the symptoms associatedwith the medical condition.

As used herein “a medical condition which may benefit from celltransplantation” refers to any medical condition which may be alleviatedby administration of the encapsulated cells of the present invention.

Examples of such medical conditions include, but are not limited to,stem cell deficiency, heart disease, neurodegenerative diseases,glaucoma neuropathy, Parkinson's disease, cancer, Schizophrenia,Alzheimer's disease, stroke, burns, loss of tissue, loss of blood,anemia, autoimmune disorders, diabetes, arthritis, graft vs. hostdisease (GvHD), neurodegenerative disorders, chronic pain, autoimmuneencephalomyelitis (EAE), systemic lupus erythematosus (SLE), rheumatoidarthritis, systemic sclerosis, Sjorgen's syndrome, multiple sclerosis(MS), Myasthenia Gravis (MG), Guillain-Barrè Syndrome (GBS), Hashimoto'sThyroiditis (HT), Graves's Disease, Insulin Dependent Diabetes Melitus(IDDM) and Inflammatory Bowel Disease.

The term or phrase “transplantation”, “cell replacement”, “implantation”or “grafting” are used interchangeably herein and refer to theintroduction of the cells of the present invention to target tissue.

As used herein the term “subject” refers to any subject (e.g., mammal),preferably a human subject.

In any of the methods described herein, the cells or media can beadministered either per se or, preferably as a part of a pharmaceuticalcomposition that further comprises a pharmaceutically acceptablecarrier.

As used herein a “pharmaceutical composition” refers to a preparation ofone or more of the chemical conjugates described herein, with otherchemical components such as pharmaceutically suitable carriers andexcipients. The purpose of a pharmaceutical composition is to facilitateadministration of a compound to a subject.

Hereinafter, the term “pharmaceutically acceptable carrier” refers to acarrier or a diluent that does not cause significant irritation to asubject and does not abrogate the biological activity and properties ofthe administered compound. Examples, without limitations, of carriersare propylene glycol, saline, emulsions and mixtures of organic solventswith water.

Herein the term “excipient” refers to an inert substance added to apharmaceutical composition to further facilitate administration of acompound. Examples, without limitation, of excipients include calciumcarbonate, calcium phosphate, various sugars and types of starch,cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

According to a preferred embodiment of the present invention, thepharmaceutical carrier is an aqueous solution of saline.

Techniques for formulation and administration of drugs may be found in“Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton, Pa.,latest edition, which is incorporated herein by reference.

One may administer the pharmaceutical composition in a systemic manner(as detailed hereinabove). Alternatively, one may administer thepharmaceutical composition locally, for example, via injection of thepharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of the present invention may be manufacturedby processes well known in the art, e.g., by means of conventionalmixing, dissolving, granulating, dragee-making, levigating, emulsifying,encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries, which facilitate processing of the active ingredients intopreparations which, can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer. For transmucosal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art.

For any preparation used in the methods of the invention, thetherapeutically effective amount or dose can be estimated initially fromin vitro and cell culture assays. Preferably, a dose is formulated in ananimal model to achieve a desired concentration or titer. Suchinformation can be used to more accurately determine useful doses inhumans.

Toxicity and therapeutic efficacy of the active ingredients describedherein can be determined by standard pharmaceutical procedures in vitro,in cell cultures or experimental animals.

The data obtained from these in vitro and cell culture assays and animalstudies can be used in formulating a range of dosage for use in human.The dosage may vary depending upon the dosage form employed and theroute of administration utilized. The exact formulation, route ofadministration and dosage can be chosen by the individual physician inview of the patient's condition, (see e.g., Fingl, et al., 1975, in “ThePharmacological Basis of Therapeutics”, Ch. 1 p. 1). For example,Parkinson's patient can be monitored symptomatically for improved motorfunctions indicating positive response to treatment.

For injection, the active ingredients of the pharmaceutical compositionmay be formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hank's solution, Ringer's solution, orphysiological salt buffer.

Dosage amount and interval may be adjusted individually to levels of theactive ingredient which are sufficient to effectively regulate theneurotransmitter synthesis by the implanted cells. Dosages necessary toachieve the desired effect will depend on individual characteristics androute of administration. Detection assays can be used to determineplasma concentrations.

Depending on the severity and responsiveness of the condition to betreated, dosing can be of a single or a plurality of administrations,with course of treatment lasting from several days to several weeks ordiminution of the disease state is achieved.

The amount of a composition to be administered will, of course, bedependent on the individual being treated, the severity of theaffliction, the manner of administration, the judgment of theprescribing physician, etc. The dosage and timing of administration willbe responsive to a careful and continuous monitoring of the individualchanging condition. For example, a treated Parkinson's patient will beadministered with an amount of cells which is sufficient to alleviatethe symptoms of the disease.

Depending on the medical condition, the subject may be administered withadditional chemical drugs (e.g., immunomodulatory, chemotherapy etc.) orcells.

Preferably the HSCs and stromal cells share some common HLA antigens.

Examples of immunosuppressive agents include, but are not limited to,methotrexate, cyclophosphamide, cyclosporine, cyclosporin A,chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine),gold salts, D-penicillamine, leflunomide, azathioprine, anakinra,infliximab (REMICADE), etanercept, TNF.alpha. blockers, a biologicalagent that targets an inflammatory cytokine, and Non-SteroidalAnti-Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are notlimited to acetyl salicylic acid, choline magnesium salicylate,diflunisal, magnesium salicylate, salsalate, sodium salicylate,diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin,ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone,phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen,Cox-2 inhibitors and tramadol.

Before explaining at least one embodiment of the invention in detail, itis to be understood that the invention is not necessarily limited in itsapplication to the details set forth in the following description orexemplified by the Examples. The invention is capable of otherembodiments or of being practiced or carried out in various ways.

As used herein the term “about” refers to ±10%.

The terms “comprises”, “comprising”, “includes”, “including”, “having”and their conjugates mean “including but not limited to”.

The term “consisting of” means “including and limited to”.

The term “consisting essentially of” means that the composition, methodor structure may include additional ingredients, steps and/or parts, butonly if the additional ingredients, steps and/or parts do not materiallyalter the basic and novel characteristics of the claimed composition,method or structure.

As used herein, the singular form “a”, “an” and “the” include pluralreferences unless the context clearly dictates otherwise. For example,the term “a compound” or “at least one compound” may include a pluralityof compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention maybe presented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to includeany cited numeral (fractional or integral) within the indicated range.The phrases “ranging/ranges between” a first indicate number and asecond indicate number and “ranging/ranges from” a first indicate number“to” a second indicate number are used herein interchangeably and aremeant to include the first and second indicated numbers and all thefractional and integral numerals therebetween.

As used herein the term “method” refers to manners, means, techniquesand procedures for accomplishing a given task including, but not limitedto, those manners, means, techniques and procedures either known to, orreadily developed from known manners, means, techniques and proceduresby practitioners of the chemical, pharmacological, biological,biochemical and medical arts.

As used herein, the term “treating” includes abrogating, substantiallyinhibiting, slowing or reversing the progression of a condition,substantially ameliorating clinical or aesthetical symptoms of acondition or substantially preventing the appearance of clinical oraesthetical symptoms of a condition.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable subcombination or as suitable in any other describedembodiment of the invention. Certain features described in the contextof various embodiments are not to be considered essential features ofthose embodiments, unless the embodiment is inoperative without thoseelements.

Various embodiments and aspects of the present invention as delineatedhereinabove and as claimed in the claims section below find experimentalsupport in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with theabove descriptions illustrate some embodiments of the invention in a nonlimiting fashion.

Generally, the nomenclature used herein and the laboratory proceduresutilized in the present invention include molecular, biochemical,microbiological and recombinant DNA techniques. Such techniques arethoroughly explained in the literature. See, for example, “MolecularCloning: A laboratory Manual” Sambrook et al., (1989); “CurrentProtocols in Molecular Biology” Volumes I-III Ausubel, R. M., ed.(1994); Ausubel et al., “Current Protocols in Molecular Biology”, JohnWiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide toMolecular Cloning”, John Wiley & Sons, New York (1988); Watson et al.,“Recombinant DNA”, Scientific American Books, New York; Birren et al.(eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, ColdSpring Harbor Laboratory Press, New York (1998); methodologies as setforth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and5,272,057; “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis,J. E., ed. (1994); “Culture of Animal Cells—A Manual of Basic Technique”by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; “Current Protocolsin Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al.(eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange,Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods inCellular Immunology”, W. H. Freeman and Co., New York (1980); availableimmunoassays are extensively described in the patent and scientificliterature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153;3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654;3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219;5,011,771 and 5,281,521; “Oligonucleotide Synthesis” Gait, M. J., ed.(1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J.,eds. (1985); “Transcription and Translation” Hames, B. D., and HigginsS. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986);“Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide toMolecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol.1-317, Academic Press; “PCR Protocols: A Guide To Methods AndApplications”, Academic Press, San Diego, Calif. (1990); Marshak et al.,“Strategies for Protein Purification and Characterization—A LaboratoryCourse Manual” CSHL Press (1996); all of which are incorporated byreference as if fully set forth herein. Other general references areprovided throughout this document. The procedures therein are believedto be well known in the art and are provided for the convenience of thereader. All the information contained therein is incorporated herein byreference.

Example 1 Materials and Methods

Cells are mixed with soluble decellularized ECM (e.g. extracted fromporcine or sheep organs) together with a polymer or hydrogel—e.g.alginate (FIG. 1).

Once the alginate-ECM-cells capsules are made, the capsules may becoated with poly-1-lysine (PLL) or chitosan. The coated microcapsulesalginate core is then liquefied using an additional step of suspensionin HEPES sodium citrate solution (or EDTA or other chelating agents).The citrate solution chelates calcium and dissolves the alginate insidethe capsule. The coating polymer network connects the alginate only(width of coat range from 10 μm to 30 μm) at the microcapsules peripheryand reduces the osmotic pressure inside the capsule. (Coated polymer canalso be connected to negative components of the ECM at the microcapsulesperiphery, in case of using positive charged polymer for themicrocapsule preparation such as chitosan).

In that way the present inventors obtained a capsule fabricated entirelyfrom ECM and coated with a coating polymer such as PLL or chitosan. At37° C. incubation, the ECM solidifies to generate a network of collagefibers inside the capsules while cells are attached or dispersed therein(alginate to ECM ratio range from 1%:99% to less than 0.1% of alginate)(FIG. 2).

Preparation of Soluble Decellularized ECM: Polymer/Hydrogel-ECM-cellencapsulation are prepared from desirable cells and nativedecellularized ECM. Native tissue is washed in PBS to remove allresidual blood. The tissue is sliced and incubated in hyper-hypo-tonicsolutions of NaCl. Slices are then incubated with trypsin-EDTA 0.05% for24 hrs (this was repeated twice). For chemical removal, tissue is thenwashed several times with triton X-100+ammonium hydroxide solutions andfinally several washing cycles of 48 hrs with PBS until no residue offoam is obtained. ECM is sterilized by washing for two hours withethanol (70%) and following two washes with double distilled water.Lyophilized ECM and protease enzyme (e.g. pepsin or/and collagenaseor/and trypsin) at different concentration and biological activities aremixed in 15 ml of 0.05 M to 0.2M HCl and kept at a constant stirring for48 h at room temperature (25° C.). The resultant viscous solution ofdigested ECM solution has a pH of approximately 3.0-4.0. The activity ofthe enzyme is irreversibly inactivated according to the type ofenzyme—for example for pepsin the pH will be raised to 7.4. Thesolubilized matrix still retains ECM proteins and peptide fragmentstherefore, the matrix retains its biochemical components, as isnecessary for cell-matrix interactions. ECM are considered completelysolubilized, when no particles are detected in solution.

Basic protocol for the Preparation of Hydrogel-ECM-cell Encapsulation:Solubilized ECM is used for cell encapsulation with alginate. Alginate(medium or low viscosity, with different G content and concentrations(NOVA MATRIX, Norway)) is mixed with solubilized ECM and cells areadded. Hydrogel-ECM-cell mixture is infused at a constant flow rate of6.7 ml/min through a 21 Gauge needle. Constant airflow of 1 psi is usedfor intersection of the mixture into droplets. Droplets of a fallingdistance of 3.5 cm is necessary to create the desired sizemicrocapsules. The hydrogel was then cross-linked with a crosslinkersuch as CaCl₂ 1.5% or strontium solutions for alginate. Coating thehydrogel spheres with PLL or chitosan or other coating polymers createsthe desirable semi permeable microcapsules. The capsules are thenliquefied using Na-citrate 1.5% solution for 5 minutes or EDTA in orderto get an ECM only based capsule surrounded by coated polymer.

Example 2

Alginate-poly-1-lysine (PLL) microcapsules were prepared with humanliver cells (Hum Hep) and human mesenchymal stem cells (hMSC). Prior toencapsulation, cells were genetically modified to express the PDX-1gene, which is a pancreatic transcription factor that regulates theexpression of multiple genes in beta cells including those that encodeinsulin. PDX-1 induces trans-differentiation of cells into cells thatsecret insulin in a glucose regulated manner. Microcapsules wereanalyzed for size, morphology, cell viability and distribution withinthe capsules.

Results

Acellular pancreatic ECM was generated. Histological analysis revealedcomplete cell removal while maintaining the ECM fibers architecture asapparent in SEM imaging (FIG. 3D). After decellularization, the ECM waslyophilized and liquefied, using proteolytic enzymes, as illustrated inFIGS. 3E-F. Proteomic analysis revealed the preservation of major ECMcomponents in both decellularized and liquefied ECM (FIG. 4A). Both thestorage modulus (G′) and the loss modulus (G″) changed over time andwere characterized after the temperature of the sample was raised from4° C. to 37° C. (FIG. 4B). G′ and G″ reached steady state afterapproximately 4-5 minutes suggesting that gelation had occurred.

Prior to the preparation of encapsulated ECM-cells, the biologicalactivity of insulin producing cells seeded on ECM gels was evaluated ascompared to those seeded on a plate. A thin layer of liquefied ECM wasspread in a 6-wells plate (FIG. 5A). Gels, made from the liquefied ECMwhich were incubated at 37° C., exhibited a 3D fiber network structuresimilar to the native tissue (FIGS. 5B-C) with a pore size andcell-fiber interaction suitable for supporting cellular proliferation(FIGS. 5D-G). Moreover, in comparison to cells seeded on a plate (FIG.5H), cells seeded on the gel exhibited a more elongated shape, probablydue to the formation of focal adhesions which connect the ECM fibers toactin filaments of the cells (FIG. 5I). The efficiency of cell seedingwas assessed by IN Cell 2000, which imaged and analyzed cell viabilityover time by analyzing Hoechst staining. C-peptide secretion of PDX-1transduced cells or non-transduced cells was evaluated. Within thepancreatic β-cell, proinsulin is cleaved into one molecule of C-peptideand one molecule of insulin. C-peptide is subsequently released into thecirculation at concentrations equimolar to those of insulin. In contrastto insulin, C-peptide is minimally extracted by the liver. Therefore,peripheral C-peptide concentration reflects the secretion of β-cell moreaccurately than insulin. As seen in FIG. 5J, the 3D culture on the geldid not affect insulin production of transfected hMSC. However, gel 3Dculture did significantly increase the insulin production of Hum Hepcells as compared to cells seeded on a plate (FIG. 5K). Thus, it can bededuced that the enclosed ECM components on 3D gels attract Hum Hepcells and contribute to insulin production.

The liquefied ECM was then incorporated with transfected liver cells orhMSCs. The solubilized ECM and cells were mixed with alginate andco-extruded through calcium and PLL solutions to produce Alginate-PLLcapsules containing both insulin producing cells and acellularpancreatic ECM.

As illustrated in FIGS. 6A-D, encapsulated cells were viable for morethan 120 days using the Alamar Blue assay and more than 108 days usingthe Fluorescein Diacetate (FDA) cell viability assay.

Therapeutic efficacy of encapsulated Hum Hep cells and hMSC secretinginsulin was performed. In the microparticles, collagen fibers were seento self-assemble into typical 3D fibrous structures inside the capsule(FIG. 7C). Moreover, cell clusters were noted to organize around largecollagen depositions originating from the liquefied ECM (FIGS. 7A, B andD). Cell encapsulation did not affect the insulin production oftransfected liver cells, although it did increase the insulin productionof encapsulated non-transfected cells (FIG. 7E). The results support thepresent hypothesis that the microenvironment inside the capsule permitsa native environment that encourages the bioactivity of these cells asglucose dependent cells even without viral transfection. However, theinsulin production of hMSCs, both transfected and untransfected, wassignificantly affected by ECM-cell encapsulation (FIGS. 7F-G). Theseresults indicate that the ECM-microenvironment within the microcapsuleis not only permissive for hMSC transdifferentiation into β-cells, butactually contributes to insulin secretion.

Although the invention has been described in conjunction with specificembodiments thereof, it is evident that many alternatives, modificationsand variations will be apparent to those skilled in the art.Accordingly, it is intended to embrace all such alternatives,modifications and variations that fall within the spirit and broad scopeof the appended claims.

All publications, patents and patent applications mentioned in thisspecification are herein incorporated in their entirety by referenceinto the specification, to the same extent as if each individualpublication, patent or patent application was specifically andindividually indicated to be incorporated herein by reference. Inaddition, citation or identification of any reference in thisapplication shall not be construed as an admission that such referenceis available as prior art to the present invention. To the extent thatsection headings are used, they should not be construed as necessarilylimiting.

What is claimed is:
 1. A microparticle which encapsulates a plurality ofcells, having a core which comprises a polymerizing agent anddecellularized extracellular matrix (ECM) of a tissue, saiddecellularized ECM comprising: continuous fibers of collagen, celladhesion polypeptides and space-filling molecules of the tissue, saiddecellularized ECM retaining structural properties of the ECM of saidtissue; and an outer layer which comprises the same polymerizing agentas said core, wherein an amount of said polymerizing agent in said outerlayer is at least 10 times the amount of said polymerizing agent in saidcore.
 2. The microparticle of claim 1, wherein said outer layer furthercomprises a polyion.
 3. The microparticle of claim 2, wherein thepolyion is selected from the group consisting of poly-L-lysine,poly-D-lysine, poly-L,D-lysine, poly-L-ornithine, poly-D-ornithine,poly-L,D ornithine, chitosan, polyacrylamide, poly(vinyl alcohol) andcombinations thereof.
 4. The microparticle of claim 1, wherein saidpolymerizing agent is selected from the group consisting of chitosan,polymethacrylic acid, a polysaccharide, poly (ethylene glycol) (PEG) andpoly (hydroxyethyl) methacrylate (HEMA).
 5. The microparticle of claim4, wherein said polysaccharide is selected from the group consisting ofalginate, hyaluronic acid and agarose.
 6. The microparticle of claim 1,wherein a thickness of said outer layer is between about 10 μm to 30 μm.7. The microparticle of claim 1, being between 300-1000 μm in diameter.8. The microparticle of claim 1, wherein said decellularizedextracellular matrix is derived from pancreatic tissue.
 9. Themicroparticle of claim 1, wherein said decellularized extracellularmatrix is derived from cardiac tissue.
 10. The microparticle of claim 8,further comprising cells which express pancreatic beta cell markers. 11.The microparticle of claim 9, further comprising cells which expresscardiac markers.
 12. A pharmaceutical composition comprising themicroparticle of claim 1 as the active agent and a pharmaceuticallyacceptable carrier.
 13. The microparticle of claim 1, wherein saidcontinuous fibers of collagen are distributed homogenously throughoutthe core and are not present as clusters or particles.
 14. Themicroparticle of claim 1 being generated by: a) decellularizing ECM of atissue under conditions that the decellularized ECM retains a structuralproperty of said ECM of said tissue; b) contacting solubilizeddecellularized ECM and cells in the presence of a polymerizing agent togenerate a mixture; (c) generating microparticles comprising said cells;and (d) depolymerization said polymerizing agent.